In the past 15 years or so, enormous progress has been made in the development of the synthesis of oligodeoxyribonucleotides (DNA sequences), oligoribonucleotides (RNA sequences) and their analogues ‘Methods in Molecular Biology, Vol. 20, Protocol for Oligonucleotides and Analogs’, Agrawal, S. Ed., Humana Press, Totowa, 1993. Much of the work has been carried out on a micromolar or even smaller scale, and automated solid phase synthesis involving monomeric phosphoramidite building blocks Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859–1862 has proved to be the most convenient approach. Indeed, high molecular weight DNA and relatively high molecular weight RNA sequences can now be prepared routinely with commercially available synthesisers. These synthetic oligonucleotides have met a number of crucial needs in biology and biotechnology.
Whereas milligram quantities have generally sufficed for molecular biological purposes, gram to greater than 100 gram quantities are required for clinical trials. Several oligonucleotide analogues that are potential antisense drugs are now in advanced clinical trials. If, as seems likely in the very near future, one of these sequences becomes approved, say, for the treatment of AIDS or a form of cancer, kilogram, multikilogram or even larger quantities of a specific sequence or sequences will be required.
Many of the oligonucleotides currently of interest in the phamaceutical industry are analogues of natural oligonucleotides which comprise phosphorothioated-internucleoside linkages. When phosphorothioate linkages are present, particularly when such linkages comprise a major proportion of the linkages, and especially when they comprise 100% of the internucleoside linkages, it is highly desirable that the concentration of impurity, non-phosphorothioated linkages in the final product is kept to a pharmacologically acceptable level.
A large number of protocols for the synthesis of oligonucleotides employ acetonitrile as a solvent for the reagents employed. Acetonitrile is attractive as a solvent because it is inert towards the reagents and oligonucleotide product, it has good solvation properties and is environmentally acceptable. Commonly, for large-scale syntheses, a high concentration of acetonitrile is present during the stage when the oligonucleotide product is cleaved from the solid support. Hitherto, this has been acceptable for large scale synthesis because of the perceived inert nature of acetonitrile. However, during the course of the studies resulting in the present invention, it has now been surprisingly found that higher purity oligonucleotides can be obtained by reducing the concentration of acetonitrile present during the cleavage stage.